GSTP1 is a member of the glutathione S-transferase enzyme superfamily, which catalyzes the conjugation of electrophiles with glutathione in the process of detoxification. GSTP1 is widely overexpressed in colorectal cancer, from aberrant crypt foci to advanced carcinomas. Increased expression of GSTP1 is associated with multidrug resistance and a worse clinical prognosis. However, GSTP1-null mice have an increased risk of tumor formation. Thus, the biological function of GSTP1 in colorectal cancer biology remains speculative. In an effort to gain further insights into the role of GSTP1 in tumorigenesis, we disrupted the GSTP1 gene in HCT116 human colorectal cancer cells using targeted homologous recombination. We find that loss of GSTP1 resulted in impaired clonogenic survival and proliferation. Specifically, under growth-limiting conditions, (a) GSTP1 protected HCT116 cells from oxidative stress and associated apoptosis and (b) promoted mitogen-activated protein kinase-extracellular signal-regulated kinase kinase/extracellular signal-regulated kinase-mediated G1-S cell cycle progression. In vivo, GSTP1 was critical for engraftment and growth of HCT116 tumor xenografts. These studies directly show that GSTP1 promotes clonogenic survival and proliferation in HCT116 human colon cancer cells.